Barbiturates are defined herein as pyrimidine derivatives in which the pyrimidine 2, 4 and 6 positions are occupied by carbonyl or thiocarbonyl radicals. Conventional radioimmunoassay methods for their determination generally employ a radiolabeled analogue of the barbiturate to be assayed, referred to hereinafter as a tracer. U.S. Pat. No. 3,766,162 discloses a barbiturate tracer in which the radioisotope .sup.14 C is substituted for an nonradioactive barbiturate carbon atom. A more complex approach comprises modifying the barbiturate to provide a convenient site for radiolabeling the barbiturate. For example, U.S. Pat. No. 3,952,091 discloses that a pendant phenolic group capable of carrying .sup.125 I may be introduced into secobarbital. The sole example given is 5-alkyl-5-[1-(p-hydroxyphenethyl-carbamyl)-2-propyl] barbituric acid which, it should be noted, is substituted with a radioiodinatable group at the 5 position of the barbiturate.
The use of such tracers in conventional radioimmunoassay procedures is well-known. For example, in the method commonly referred to as a competitive radioimmunoassay the tracer is allowed to compete with any test substance in the sample for a limited amount of antibody capable of binding both the tracer and the test substance. The antibody-bound tracer and test substance are then separated from the unbound material by conventional methods such as precipitation with a second antibody, antibody absorption on an insolubilized surface or the charcoal separation technique disclosed by Herbert et al., J. Clin. Endocr. 25, 1375-1384 (1965). The distribution of radioactivity between the two fractions is then determined. In the case of an assay for barbiturate, a surfeit of barbiturate in the test sample will result in a proportionately large number of antibody binding sites being occupied with nonradioactive barbiturate from the sample. On the other hand a sample largely devoid of barbiturate will ineffectively compete for the antibody binding sites, thereby resulting in proportionately high radioactivity in the bound phase. Other related radioimmunoassay methods which may be used with barbiturate tracers include the solid phase or double antibody separation techniques.
Tracers should exhibit certain characteristics for optimal use in radioimmunoassays. These include high specific tracer activity and high avidity of the antibody for the tracer and sample barbiturate. In the case of specific activity, each mole of tracer should emit as high a level of radioactivity as is commensurate with reagent stability and immunoreactivity. Thus, the higher the radioactivity emitted by the tracer the lower its detection limit becomes, and this in turn increases the sensitivity of the assay.
Antibodies should bind tracers with equally high avidity as the sample barbiturate to which the assay is directed. A large tracer substituent may inhibit binding of the tracer by antibody, particularly if the substituent is in the determinant region of the sample barbiturate. The consequence of this inhibition will be an assay indicating an erroneously high concentration of barbiturate.
Prior art barbiturate radioimmunoassays which employ radioiodinated moieties substituted at the 5 position of the pyrimidine ring are largely incapable of distinguishing among barbiturates or their metabolic products which differ from one another only at this position. This includes such barbiturates as barbital, phenobarbital, propallylonal and secobarbital as well as metabolites such as p-hydroxyphenobarbital. See, for example, "Clinical Chemistry," Vol. 23, No. 5, pages 873-876 (1977) in which it is reported that a barbiturate radioimmunoassay kit containing an .sup.125 I-labeled secobarbital derivative is subject to interference by p-hydroxyphenobarbital.
It is therefore a general object of this invention to provide new barbiturate derivatives which are useful in the preparation of tracers for the radioimmunoassay of barbiturates and which may be radioiodinated to provide the tracers themselves.
In particular, it is an object of this invention to obtain a barbiturate tracer which can be used in a radioimmunoassay capable of distinguishing among the various barbiturates of medical interest as well as their metabolites.
It is an additional object of this invention to prepare a tracer which will bind antibody with an avidity close to that of the barbiturate which is being assayed.
It is a further object of this invention to provide a tracer having high specific activity.
These and other objects of this invention will be apparent to those skilled in the art from a consideration of this specification taken in its entirety.